Method for determining content of glutathione in bacterial cells

A technology for glutathione content and determination method, which is applied in the field of determination of intracellular glutathione content of Gram-positive bacteria, can solve the problems of low results, cumbersome operation, high cost of equipment and consumables, and achieves low cost, measurement Accurate, easy-to-use results

Active Publication Date: 2014-02-19
SICHUAN UNIV
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  • Abstract
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Problems solved by technology

For Gram-positive bacteria, methods such as ultrasonic wall breaking and liquid nitrogen grinding cannot completely break the wall, so intracellular glutathione cannot be completely detected; lysozyme will destroy glutathione, resulting in low results
Although some methods are accurate in measurement, the operation is cumbersome, and the cost of equipment and consumables is extremely high.

Method used

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  • Method for determining content of glutathione in bacterial cells
  • Method for determining content of glutathione in bacterial cells
  • Method for determining content of glutathione in bacterial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] In this embodiment, the intracellular glutathione content of Streptococcus mutans is measured, and the specific method is as follows.

[0074] 1. Establish a standard curve

[0075] 1.1 Solution preparation: Prepare pH 8.0 PBS buffer solution; mBBr is made into 10mmol / L mother solution with DMSO, and stored at -20°C in the dark for 1 month, avoiding repeated freezing and thawing, and the working concentration is 100 μmol / L.

[0076] 1.2 Prepare glutathione with PBS buffer solution to make standard solutions with final concentrations of 0, 1, 2, 5, 10, and 20 μmol / L respectively, and add 2 μL of mBBr reagent to each gradient solution. The reaction system is shown in Table 1:

[0077] Table 1. Preparation of glutathione standard

[0078]

[0079] 1.3 React glutathione solutions with different concentrations prepared in step B at 37°C for 30 min for detection of fluorescence intensity.

[0080] 1.4 Draw a standard curve of different concentrations of glutathione and ...

Embodiment 2

[0115] This example is for the determination of intracellular glutathione content of Streptococcus mutans and gshAB mutant strains under different culture conditions.

[0116] Streptococcus mutans and glutathione synthase-deficient mutant strain (△gshAB) were recovered and subcultured, and the second generation was selected to inoculate in BHI liquid medium and CDM (glutathione-lacking) medium. at 80%N 2 , 20%CO 2 Incubate in an anaerobic environment at 37°C for 15 hours. After the smear test shows pure culture, 1) count the plate; 2) centrifuge 30ml of the bacterial solution at 4,000×g for 10min at 4°C to collect the bacteria.

[0117] The intracellular glutathione content of Streptococcus mutans and the △gshAB mutant strain was determined by the same mBBr plate counting method as in Example 1, and the determination results are shown in Table 4.

[0118]There are two ways for Streptococcus mutans to obtain glutathione, synthesis and ingestion. CDM is a glutathione-deficien...

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Abstract

The invention discloses a method for determining the content of glutathione in bacterial cells. The method comprises the following steps: drafting a standard curve of different concentrations of glutathione to the fluorescence intensity; adopting a plate colony-counting method to carry out colony counting; setting a bacterial liquid experiment group for determination and a background fluorescence control group; processing the experiment group and the control group, and detecting by adopting a fluorescence intensity detection method; calculating according to the standard curve to obtain the glutathione content, and calculating according to flat counting to obtain the quantity of sample bacteria; and calculating a ratio of the glutathione content to the quantity of the corresponding bacteria to obtain the content of glutathione in a certain quantity of bacterial cells. An mBBr reagent is adopted in the invention, can go through the cell walls of Gram-positive bacteria, enters the cells, and combines with glutathione to generate a stable fluorescence derivative, and the bacteria are cracked by acetonitrile, so a problem of the wall breaking of the Gram-positive bacteria of the measurement method is solved.

Description

technical field [0001] The invention relates to a method for detecting glutathione, in particular to a method for determining intracellular glutathione content of Gram-positive bacteria. Background technique [0002] Glutathione is a small molecule tripeptide, as an important redox signaling molecule, widely exists in eukaryotic cells. At the same time, glutathione is also the most important non-protein sulfhydryl compound in microbial cells except archaea, and its effect on microorganisms remains to be further studied. The determination of glutathione content in bacteria helps to understand the source and destination of glutathione. [0003] At present, there are various methods for the determination of glutathione, and they are mainly concentrated in samples such as eukaryotic cells and body fluids. The determination method of glutathione has been continuously improved. There are mainly spectrophotometry, fluorescence photometry, electrochemical method and so on. With ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N1/28
Inventor 李雨庆徐欣刘向红李明云郭强周学东
Owner SICHUAN UNIV
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